The second messengers, adenosine 3′,5′ cyclic monophosphate (cAMP) and guanosine 3′,5′ cyclic monophosphate (cGMP), are important intracellular mediators of a variety of cellular functions including cell growth, differentiation, apoptosis, and cell death. Production of cAMP is controlled through the adenylyl cyclase family of enzymes, which convert adenosine triphosphate (ATP) to cAMP and inorganic pyrophosphate (PPi). The adenylyl cyclases are activated or inhibited via direct interaction with membrane bound G-protein coupled receptor (GPCR) α-subunits. When an α-subunit of a stimulatory GPCR is activated, designated Gαs, adenylyl cyclase converts ATP to cAMP and PPi. Conversely, when an α-subunit of an inhibitory GPCR is activated, designated Gαi, an inhibitory effect on adenylyl cyclase is exerted and the conversion of ATP to cAMP and PPi is not realized. G-protein coupled receptors play a prominent role in a wide variety of biological processes such as neurotransmission, cardiac output, and pain modulation. Their importance in developing new medically useful compounds is well understood; as such they are highly targeted in drug discovery research.
The intracellular concentration of cAMP is also affected by another group of enzymes, cyclic nucleotide phosphodiesterases (PDE), which catalyze the hydrolysis of cAMP to AMP and cyclic cGMP to GMP. Phosphodiesterases function in conjunction with adenylyl cyclases and guanylate cyclases to regulate the amplitude and duration of cell signaling mechanisms that are mediated by cAMP and cGMP. Phosphodiesterases therefore regulate a wide range of important biological responses to first messengers such as hormones, light, and neurotransmitters. There are two classes of PDEs; Class I are found in the cytoplasm or bound to intracellular organelles or membranes of all eukaryotic cells, whereas Class II PDEs are not well characterized and have only been found in lower eukayotes. Cellular responses controlled by Class I phosphodiesterases, through control of cAMP and cGMP conversion, include neuronal responses, aldosterone production, regulation of platelet aggregation, insulin regulation, emesis, regulation of smooth muscle tension, visual phototransduction, and modulation of T-cell responsiveness. Numerous clinically important compounds are known to inhibit phosphodiesterases including; rolipram, theophylline, and sildenafil. Therefore, inhibitors of phosphosdiesterases are also important targets in drug discovery.
The second messenger cAMP is known to activate cAMP dependent protein kinase (PKA). Mammalian holo-PKA is a tetramer, made up of two regulatory and two catalytic subunits. cAMP binds to the regulatory subunits, thereby dissociating holo-PKA into its catalytic and regulatory subunits. Once released, the free catalytic subunits are capable of phosphorylating a multitude of cellular proteins, thereby causing changes in cellular functions such as muscle contraction, activation of cell cycle, activation of transcriptional activity, and DNA processing.
Because the activation or inhibition of GPCR and subsequent activation or inhibition of adenylyl cyclase results in an increase or decrease in intracellular cAMP, agents that affect their activity are important targets for drug discovery. Drugs that target GPCR account for many of the medicines sold worldwide due to the tremendous variety of biological processes relating to G-protein coupled receptors. Examples of drugs that influence GPCR include Claritin® and Alayert® (loratadine) which are used for relieving allergy symptoms, Paxil® (paroxetine HCl) for relief of depression, and Vasotec® (enalapril maleate) for relief of hypertension. Because of their importance, various GPCR assays have been developed to determine the effect of agonists and antagonists on these system components, mainly by assaying for the increase or decrease in cAMP levels. Limitations of these methods include non-homogeneous assays that require multiple dispensing steps, long incubation times, and the need for expensive equipment.
Therefore, what are needed are assays that require less manipulation than currently available technologies (e.g. two steps or less), assays that provide shorter incubation times (e.g., less than 1 hour), and assays that utilize low cost equipment while maintaining high throughput system (HTS) capabilities (e.g., luminescent based equipment). Such streamlining and cost effectiveness will allow for faster and easier evaluation of targets for drug discovery. Furthermore, luminescent based assays are not prone to interference from fluorescence; that is useful in screening large libraries of chemicals to discover the next potential drug.